Upconversion of light for use in optogenetic methods

ABSTRACT

Provided herein are compositions comprising lanthanide-doped nanoparticles which upconvert electromagnetic radiation from infrared or near infrared wavelengths into the visible light spectrum. Also provided herein are methods activating light-responsive opsin proteins expressed on plasma membranes of neurons and selectively altering the membrane polarization state of the neurons using the light delivered by the lanthanide-doped nanoparticles.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.13/882,703 filed Apr. 30, 2013, which is a national stage filing under35 U.S.C. § 371 of PCT/US2011/059287, filed Nov. 4, 2011, which claimspriority to U.S. Provisional Application No. 61/410,729, filed Nov. 5,2010, each of which applications is incorporated herein by reference inits entirety.

FIELD OF THE INVENTION

This application pertains to compositions comprising lanthanide-dopednanoparticles which upconvert electromagnetic radiation from infrared ornear infrared wavelengths into the visible light spectrum and methods ofusing lanthanide-doped nanoparticles to deliver light to activatelight-responsive opsin proteins expressed in neurons and selectivelyalter the membrane polarization state of the neurons.

BACKGROUND

Optogenetics is the combination of genetic and optical methods used tocontrol specific events in targeted cells of living tissue, even withinfreely moving mammals and other animals, with the temporal precision(millisecond-timescale) needed to keep pace with functioning intactbiological systems. The hallmark of optogenetics is the introduction offast light-responsive opsin channel or pump proteins to the plasmamembranes of target neuronal cells that allow temporally precisemanipulation of neuronal membrane potential while maintaining cell-typeresolution through the use of specific targeting mechanisms. Among themicrobial opsins which can be used to investigate the function of neuralsystems are the halorhodopsins (NpHRs), used to promote membranehyperpolarization when illuminated, and the channelrhodopsins, used todepolarize membranes upon exposure to light. In just a few short years,the field of optogenetics has furthered the fundamental scientificunderstanding of how specific cell types contribute to the function ofbiological tissues, such as neural circuits, in vivo. Moreover, on theclinical side, optogenetics-driven research has led to insights into theneurological mechanisms underlying complex mammalian behaviors such asanxiety, memory, fear, and addiction.

In spite of these advances, use of optogenetic methods in animalssuffers from the significant drawback of requiring the animal to eitherbe tethered to a light source or to have a light source surgicallyimplanted into the animal. Moreover, when optogenetic methods are usedto alter the function of neurons in the brain, a light source must beplaced in proximity to those neurons. This requires drilling a hole inthe animal's skull and also presents practical difficulties when thebrain region of interest is located deep within the brain itself. Sincelight poorly passes through neural tissue, this necessitates inserting afiber optic light source into the brain, which can result in unintendeddamage to surrounding brain tissue.

What is needed, therefore, is a method to non-invasively deliver lightto neurons located within the brain and the peripheral nervous system ofanimals expressing light-responsive opsin proteins on the plasmamembranes of neural cells.

Throughout this specification, references are made to publications(e.g., scientific articles), patent applications, patents, etc., all ofwhich are herein incorporated by reference in their entirety.

SUMMARY OF THE INVENTION

Provided herein are compositions and methods for non-invasivelydelivering light to neurons expressing light-responsive opsin proteinson neural plasma membranes via the use of nanoparticles capable ofupshifting electromagnetic radiation from wavelengths associated withthe infrared (IR) or near infrared (NIR) spectrum into wavelengthsassociated with visible light.

Accordingly, provided herein is a method to depolarize the plasmamembrane of a neural cell in an individual comprising: (a) placing aplurality of lanthanide-doped nanoparticles in proximity to the neuralcell; and (b) exposing the plurality of nanoparticles to electromagneticradiation in the infrared (IR) or near infrared (NIR) spectrum, whereinthe electromagnetic radiation in the IR or NIR spectrum is upconvertedinto light in the visible spectrum by the nanoparticles, and wherein alight-responsive opsin is expressed on the plasma membrane of the neuralcells and activation of the opsin by the light in the visible spectruminduces the depolarization of the plasma membrane.

In other aspects, provided herein is a method to depolarize the plasmamembrane of a neural cell in an individual comprising: (a) administeringa polynucleotide encoding a light-responsive opsin to an individual,wherein the light-responsive protein is expressed on the plasma membraneof a neural cell in the individual, and the opsin is capable of inducingmembrane depolarization of the neural cell when illuminated with light;(b) administering a plurality of lanthanide-doped nanoparticles inproximity to the neural cell; and (c) exposing the plurality ofnanoparticles to electromagnetic radiation in the infrared (IR) or nearinfrared (NIR) spectrum, wherein the electromagnetic radiation in the IRor NIR spectrum is upconverted into light in the visible spectrum andthe activation of the opsin by the light in the visible spectrum inducesthe depolarization of the plasma membrane.

In some aspects, provided herein is a method to hyperpolarize the plasmamembrane of a neural cell in an individual comprising: (a) placing aplurality of lanthanide-doped nanoparticles in proximity to the neuralcell; and (b) exposing the plurality of nanoparticles to electromagneticradiation in the infrared (IR) or near infrared (NIR) spectrum, whereinthe electromagnetic radiation in the IR or NIR spectrum is upconvertedinto light in the visible spectrum by the nanoparticles, and wherein alight-responsive opsin is expressed on the plasma membrane andactivation of the opsin by the light in the visible spectrum induces thehyperpolarization of the plasma membrane.

In yet other aspects, provided herein is a method to hyperpolarize theplasma membrane of a neural cell in an individual comprising: (a)administering a polynucleotide encoding a light-responsive opsin to anindividual, wherein the light-responsive protein is expressed on theplasma membrane of a neural cell in the individual, and the opsin iscapable of inducing membrane hyperpolarization of the neural cell whenilluminated with light; (b) administering a plurality oflanthanide-doped nanoparticles in proximity to the neural cell; and (c)exposing the plurality of nanoparticles to electromagnetic radiation inthe infrared (IR) or near infrared (NIR) spectrum, wherein theelectromagnetic radiation in the IR or NIR spectrum is upconverted intolight in the visible spectrum and the activation of the opsin by thelight in the visible spectrum induces the hyperpolarization of theplasma membrane.

The present disclosure is directed to apparatuses and methods involvingupconversion for deep delivery of light in vivo. Aspects of the presentdisclosure relate generally to delivery of light to tissue in vivo usingupconversion of near infrared light to the visible light spectrum andmethods relating to the applications discussed herein.

Certain aspects of the present disclosure are directed to a light sourcethat is implanted within living tissue. Nanoparticles from thenanoparticle solution anchor to a target cell population that includescells expressing light responsive channels/opsins. The nanoparticles areconfigured to respond to receipt of light of a first wavelength byemitting light of a second, different wavelength. For example, thenanoparticles can upconvert received light and thereby emit light of ahigher frequency.

Embodiments of the present disclosure are directed towards injection ofa site of interest with a virus, caring an opsin gene and a nanoparticlesolution. The virus causes a target cell population at the site ofinterest to express the opsin gene. Various different light sources arepossible. The use of different wavelengths can be particularly usefulfor facilitating the use of different (external) light sources, e.g., ascertain wavelengths exhibit corresponding decreases in absorption bytissue of the brain or otherwise.

Consistent with a particular embodiment of the present disclosure, alight-emitting diode (“LED”) is placed on a portion of a skull that hasbeen thinned. The LED is placed under the skin near the thinned portionof the skull, and the location and/or orientation of the LED is chosen,at least in part, based on the location of the target cell population.For example, the LED can be placed to reduce the distance between theLED and the target cell population and oriented accordingly.

In certain more specific aspects of the present disclosure, light fromthe LED travels through surrounding tissue to the nanoparticles. When(near) infrared light hits the nanoparticles, the nanoparticles absorbthe infrared (IR) photons and emit visible photons. The visible photonsare then absorbed by the opsins expressed within the target cellpopulation causing a response therein (e.g., triggering neuralexcitation or inhibition).

The LED can be powered by a battery similar to those used forpacemakers. The LED can emit light in the infrared spectrum, andparticularly between 700 nm-1000 nm, which can travel through the skulland intervening tissue. The light emitted from the nanoparticles has aspectra centered between 450-550 nm. The wavelength of the light emittedis dependent on characteristics of the nanoparticle.

The above overview is not intended to describe each illustratedembodiment or every implementation of the present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

Various example embodiments may be more completely understood inconsideration of the following description and the accompanyingdrawings, in which:

FIG. 1 shows a cross section of a skull, consistent with an embodimentof the present disclosure.

FIG. 2 shows light delivery to target neurons, consistent with anembodiment of the present disclosure.

FIG. 3 depicts a system that uses multiple light sources, consistentwith an embodiment of the present disclosure.

While the present disclosure is amenable to various modifications andalternative forms, specifics thereof have been shown by way of examplein the drawings and will be described in detail. It should beunderstood, however, that the intention is not to limit the presentdisclosure to the particular embodiments described. On the contrary, theintention is to cover all modifications, equivalents, and alternativesfalling within the scope of the present disclosure including aspectsdefined in the claims.

DETAILED DESCRIPTION

This invention provides, inter alia, compositions and methods fordelivering light to neural cells expressing one or more light-responsiveopsin proteins on the plasma membranes of those neural cells. Theinventors have discovered that nanoparticles doped with a lanthanidemetal (for example, Gadolinium) that converts infrared (IR) or nearinfrared (NIR) electromagnetic radiation into wavelengths correspondingto the visible light spectrum can be used to activate light-responsiveopsin proteins on the plasma membrane of a neural cell and selectivelyalter the membrane polarization state of the cell. Unlike visible light,IR or NIR electromagnetic energy readily penetrates biological tissues.For example, NIR can penetrate biological tissues for distances of up to4 centimeters (Heyward & Dale Wagner, “Applied Body CompositionAssessment”, 2nd edition (2004), p. 100). Certain equations useful forcalculating light penetration in tissue as a function of wavelength aredisclosed in U.S. Pat. No. 7,043,287, the contents of which areincorporated herein by reference. Similarly, U.S. Patent ApplicationPublication No. 2007/0027411 discloses that near infrared Low LevelLaser Treatment light penetrates the body to a depth of between 3-5 cm.Therefore, use of IR or NIR sources of electromagnetic radiation inoptogenetic methods can alleviate the need to place a light source indirect proximity to neural cells. In particular, for optogenetictechniques in the brain, use of lanthanide-doped nanoparticles incombination with IR or NIR electromagnetic energy can permit activationof the opsin protein without the need to puncture the skull or insert afiber optic light source into the brain. Similarly, in the peripheralnervous system, opsin-expressing nerves can be activated via IR or NIRsources placed under the skin or worn against the skin.

General Techniques

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology, microbiology,cell biology, biochemistry, nucleic acid chemistry, immunology, andphysiology, which are well known to those skilled in the art. Suchtechniques are explained fully in the literature, such as, MolecularCloning: A Laboratory Manual, second edition (Sambrook et al., 1989) andMolecular Cloning: A Laboratory Manual, third edition (Sambrook andRussel, 2001), (jointly referred to herein as “Sambrook”); CurrentProtocols in Molecular Biology (F. M. Ausubel et al., eds., 1987,including supplements through 2001); PCR: The Polymerase Chain Reaction,(Mullis et al., eds., 1994); Harlow and Lane (1988), Antibodies, ALaboratory Manual, Cold Spring Harbor Publications, New York; Harlow andLane (1999), Using Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (jointly referred to hereinas “Harlow and Lane”), Beaucage et al. eds., Current Protocols inNucleic Acid Chemistry, John Wiley & Sons, Inc., New York, 2000),Handbook of Experimental Immunology, 4th edition (D. M. Weir & C. C.Blackwell, eds., Blackwell Science Inc., 1987), and Gene TransferVectors for Mammalian Cells (J. M. Miller & M. P. Calos, eds., 1987).Other useful references include Harrison's Principles of InternalMedicine (McGraw Hill; J. Isseleacher et al., eds.) and LanthanideLuminescence: Photophysical, Analytical and Biological Aspects(Springer-Verlag, Berlin, Heidelberg; Hanninen & Harma, eds., 2011).

Definitions

As used herein, “infrared” or “near infrared” or “infrared light” or“near infrared light” refers to electromagnetic radiation in thespectrum immediately above that of visible light, measured from thenominal edge of visible red light at 0.74 μm, and extending to 300 μm.These wavelengths correspond to a frequency range of approximately 1 to400 THz. In particular, “near infrared” or “near infrared light” alsorefers to electromagnetic radiation measuring 0.75-1.4 μm in wavelength,defined by the water absorption.

“Visible light” is defined as electromagnetic radiation with wavelengthsbetween 380 nm and 750 nm. In general, “electromagnetic radiation,”including light, is generated by the acceleration and deceleration orchanges in movement (vibration) of electrically charged particles, suchas parts of molecules (or adjacent atoms) with high thermal energy, orelectrons in atoms (or molecules).

The term “nanoparticles” as used herein, can also refer to nanocrystals,nanorods, nanoclusters, clusters, particles, dots, quantum dots, smallparticles, and nanostructured materials. The term “nanoparticle”encompasses all materials with small size (generally, though notnecessarily) less than 100 nm associated with quantum size effects.

An “individual” is a mammal including a human. Mammals include, but arenot limited to, farm animals, sport animals, pets, primates, mice andrats. Individuals also include companion animals including, but notlimited to, dogs and cats. In some aspects, an individual is a non-humananimal, such as a mammal. In another aspect, an individual is a human.

As used herein, the singular form “a”, “an”, and “the” includes pluralreferences unless indicated otherwise.

It is intended that every maximum numerical limitation given throughoutthis specification includes every lower numerical limitation, as if suchlower numerical limitations were expressly written herein. Every minimumnumerical limitation given throughout this specification will includeevery higher numerical limitation, as if such higher numericallimitations were expressly written herein. Every numerical range giventhroughout this specification will include every narrower numericalrange that falls within such broader numerical range, as if suchnarrower numerical ranges were all expressly written herein.

Lanthanide-Doped Nanoparticles

In materials science, doping is commonly used to incorporate specificspecies of ions or atoms into a host lattice core structure to producehybrid materials with new and useful properties. When synthesizingnanoparticles, doping can influence not only the size and shape of theparticles, but also other properties, such as the ability to convertnear infrared (NIR) excitation into a visible emission of light.

The lanthanide metals, or lanthanoids (also known as the “Rare Earth”metals), are elements of atomic number 57 (Lanthanum) through 71(Lutetium), and often include Yttrium (atomic number 39) and Scandium(atomic number 21) because of their chemical similarities. Lanthanideions exhibit unique luminescent properties, including the ability toconvert near infrared long-wavelength excitation radiation into shortervisible wavelengths through a process known as photon upconversion.Lanthanides usually exist as trivalent cations, in which case theirelectronic configuration is (Xe) 4f, with n varying from 1 (Ce³⁺) to 14(Lu³⁺). The transitions within the f-manifold are responsible for manyof the photo-physical properties of the lanthanide ions, such aslong-lived luminescence and sharp absorption and emission lines. Thef-electrons are shielded from external perturbations by filled 5s and 5porbitals, thus giving rise to line-like spectra. Additionally, the f-felectronic transitions of lanthanides are LaPorte forbidden, leading tolong excited state lifetimes, in the micro- to millisecond range.

In some embodiments, any known method can be used to synthesizelanthanide-doped nanoparticles. Such methods are well known in the art(See, e.g., Xu & Li, 2007, Clin Chem., 53(8):1503-10; Wang et al., 2010,Nature, 463(7284):1061-5; U.S. Patent Application Publication Nos.:2003/0030067 and 2010/0261263; and U.S. Pat. No. 7,550,201, thedisclosures of each of which are incorporated herein by reference intheir entireties). For example, in some embodiments, lanthanide-dopednanorods can be synthesized with a NaYF₄ dielectric core, wherein a DIwater solution (1.5 ml) of 0.3 g NaOH is mixed with 5 ml of ethanol and5 ml of oleic acid under stirring. To the resulting mixture isselectively added 2 ml of RECl₃ (0.2 M, RE=Y, Yb, Er, Gd, Sm, Nd or La)and 1 ml of NH₄F (2 M). The solution is then transferred into anautoclave and heated at 200° C. for 2 h. Nanorods are then obtained bycentrifugation, washed with water and ethanol several times, and finallyre-dispersed in cyclohexane. In another non-limiting example,nanoparticles can be synthesized using 2 ml of RECl₃ (0.2 M, RE=Y, Yb,Er, Gd, or Tm) in methanol added to a flask containing 3 ml oleic acidand 7 ml of 1-octadecene. This solution is then heated to 160° C. for 30min and cooled down to room temperature. Thereafter, a 5 ml methanolsolution of NH₄F (1.6 mmol) and NaOH (1 mmol) is added and the solutionis stirred for 30 min. After methanol evaporation, the solution is nextheated to 300° C. under argon for 1.5 h and cooled down to roomtemperature. The resulting nanoparticles are precipitated by theaddition of ethanol, collected by centrifugation, washed with methanoland ethanol several times, and finally re-dispersed in cyclohexane.

In one embodiment, the materials for the lanthanide-doped nanoparticlecore can include a wide variety of dielectric materials. In variousembodiments, the dielectric core can include lanthanide-doped oxidematerials. Lanthanides include lanthanum (La), cerium (Ce), praseodymium(Pr), neodymium (Nd), promethium (Pm), samarium (Sm), europium (Eu),gadolinium (Gd), terbium (Tb), dysprosium (Dy), holmium (Ho), erbium(Er), thulium (Tm), ytterbium (Yb), and lutetium (Lu). Other suitabledielectric core materials include non-lanthanide elements such asyttrium (Y) and scandium (Sc). Hence, suitable dielectric core materialsinclude, but are not limited to, Y₂O₃, Y₂O₂S, NaYF₄, NaYbF4, Na dopedYbF₃, YAG, YAP, Nd₂O₃, LaF₃, LaCl₃, La₂O₃, TiO₂, LuPO₄, YVO₄, YbF₃, YF₃,or SiO₂. In one embodiment, the dielectric nanoparticle core is NaYF₄.These dielectric cores can be doped with one or more Er, Eu, Yb, Tm, Nd,Tb, Ce, Y, U, Pr, La, Gd and other rare-earth species or a combinationthereof. In one embodiment, the dielectric core material is doped withGd. In another embodiment, the lanthanide-doped nanoparticle comprisesNaYF₄:Yb/X/Gd, wherein X is Er, Tm, or Er/Tm. In some embodiments, thelanthanide-doped nanoparticles comprise a NaYF₄:Yb/Er (18/2 mol %)dielectric core doped with any of about 0 mol %, about 5 mol %, about 10mol %, about 15 mol %, about 20 mol %, about 25 mol %, about 30 mol %,about 35 mol %, about 40 mol %, about 45 mol %, about 50 mol %, about 55mol %, about or 60 mol % Gd³⁺ ions, inclusive, including any mol % inbetween these values. In other embodiments, the lanthanide-dopednanoparticles comprise a NaYF₄:Yb/Er (18/2 mol %) dielectric core dopedwith any of about 0 mol %, about 5 mol %, about 10 mol %, about 15 mol%, about 20 mol %, about 25 mol %, or about 30 mol % Yb³⁺ ions,inclusive, including any mol % in between these values. In yet otherembodiments, the lanthanide-doped nanoparticles comprise a NaYF₄:Yb/Er(18/2 mol %) dielectric core doped with any of about 0 mol %, about 5mol %, about 10 mol %, about 15 mol %, about 20 mol %, about 25 mol %,or about 30 mol % Er³⁺ ions, inclusive, including any mol % in betweenthese values. In other embodiments, the lanthanide-doped nanoparticlescomprise a NaYF₄:Yb/Er (18/2 mol %) dielectric core doped with any ofabout 0 mol %, about 5 mol %, about 10 mol %, about 15 mol %, about 20mol %, about 25 mol %, or about 30 mol % Tm³⁺ ions, inclusive, includingany mol % in between these values. In another embodiment, thelanthanide-doped nanoparticle is selected from the group consisting ofNaYF₄:Yb/Er/Gd (18/2/5 mol %), NaYF₄:Yb/Tm/Er/Gd (20/0.2/0.1/5 mol %),NaYF₄:Yb/Tm/Er/Gd (20/0.2/0.05/5 mol %), and NaYF₄:Yb/Tm/Gd (20/0.2/5mol %).

In some aspects, the lanthanide-doped nanoparticles disclosed herein areconjugated to one or more delivery molecules to target them to one ormore molecules expressed on the surface of a neural cell of interest(such as a neural cell expressing one or more light-responsive opsinproteins on its plasma membrane). These can include, without limitation,antibodies or fragments thereof, small molecules, as well as lectins orany other carbohydrate motif. The delivery molecules ensure that thelanthanide-doped nanoparticles remain in close proximity to the opsinproteins to permit activation upon upconversion of IR or NIRelectromagnetic radiation. Antibody conjugation to nanoparticles iswell-known in the art (See, e.g., U.S. Patent Application PublicationNo.: 2010/0209352 and 2008/0267876, the contents of each of which areincorporated by reference herein in their entireties).

In another aspect, lanthanide-doped nanoparticles can be embedded ortrapped within a biocompatible material which is surgically placedproximal to (such as adjacent to or around) the neural cell of interest(such as a neural cell expressing one or more light-responsive opsinproteins on its plasma membrane). In some embodiments, the biocompatiblematerial is transparent, so that visible light produced by theupconversion of IR or NIR electromagnetic radiation by thelanthanide-doped nanoparticles can reach the light-responsive opsinproteins expressed on the surface of the neural cell of interest. Thebiocompatible materials used to embed or trap the lanthanide-dopednanoparticles can include, but are not limited to, Ioplex materials andother hydrogels such as those based on 2-hydroxyethyl methacrylate oracrylamide, and poly ether polyurethane ureas (PEUU) including Biomer(Ethicon Corp.), Avcothane (Avco-Everrett Laboratories), polyethylene,polypropylene, polytetrafluoroethylene (Gore-Tex), poly(vinylchloride),polydimethylsiloxane, an ethylene-acrylic acid copolymer, knitted orwoven Dacron, polyester-polyurethane, polyurethane,polycarbonatepolyurethane (Corethane), polyamide (Nylon) andpolystyrene. In one embodiment, the biocompatible material can bepolydimethylsiloxane (PDMS). Additional compounds that may be used forembedding and/or trapping the lanthanide-doped nanoparticles disclosedherein are described in Kirk-Othmer, Encyclopedia of ChemicalTechnology, 3rd Edition 1982 (Vol. 19, pp. 275-313, and Vol. 18, pp.219-2220), van der Giessen et al., 1996, Circulation, 94:1690-1997(1996), U.S. Patent Application Publication No.: 2011/0054305, and U.S.Pat. No. 6,491,965, the contents of each which are incorporated hereinby reference in their entireties.

Light-Responsive Opsin Proteins

Provided herein are optogenetic-based compositions for selectivelyhyperpolarizing or depolarizing neurons of the central or peripheralnervous system. Optogenetics refers to the combination of genetic andoptical methods used to control specific events in targeted cells ofliving tissue, even within freely moving mammals and other animals, withthe temporal precision (millisecond-timescale) needed to keep pace withfunctioning intact biological systems. Optogenetics requires theintroduction of fast light-responsive channel or pump proteins to theplasma membranes of target neuronal cells that allow temporally precisemanipulation of neuronal membrane potential while maintaining cell-typeresolution through the use of specific targeting mechanisms.

Light-responsive opsins that may be used in the present inventioninclude opsins that induce hyperpolarization in neurons by light andopsins that induce depolarization in neurons by light. Examples ofopsins are shown in Tables 1 and 2 below.

Table 1 shows identified opsins for inhibition of cellular acitivtyacross the visible spectrum:

Wavelength Opsin Type Biological Origin Sensitivity Defined action NpHRNatronomonas 589 nm max Inhibition pharaonis (hyperpolarization) BRHalobacterium 570 nm max Inhibition helobium (hyperpolarization) ARAcetabulaira 518 nm max Inhibition acetabulum (hyperpolarization) GtR3Guillardia theta 472 nm max Inhibition (hyperpolarization) MacLeptosphaeria 470-500 nm max Inhibition maculans (hyperpolarization)NpHr3.0 Natronomonas 680 nm utility Inhibition pharaonis 589 nm max(hyperpolarization) NpHR3.1 Natronomonas 680 nm utility Inhibitionpharaonis 589 nm max (hyperpolarization)Table 2 show identified opsins for excitation and modulation across thevisible specturm:

Wavelength Opsin Type Biological Origin Sensitivity Defined action VChR1Volvox carteri 589 nm utility Excitation 535 nm max (depolarization)DChR Dunaliella salina 500 nm max Excitation (depolarization) ChR2Chlamydomonas 470 nm max Excitation reinhardtii 380-405 nm utility(depolarization) ChETA Chlamydomonas 470 nm max Excitation reinhardtii380-405 nm utility (depolarization) SFO Chlamydomonas 470 nm maxExcitation reinhardtii 530 nm max (depolarization) Inactivation SSFOChlamydomonas 445 nm max Step-like reinhardtii 590 nm; activation390-400 nm (depolarization) Inactivation C1V1 Volvox carteri and 542 nmmax Excitation Chlamydomonas (depolarization) reinhardtii C1V1 E122Volvox carteri and 546 nm max Excitation Chlamydomonas (depolarization)reinhardtii C1V1 E162 Volvox carteri and 542 nm max ExcitationChlamydomonas (depolarization) reinhardtii C1V1 Volvox carteri and 546nm max Excitation E122/E162 Chlamydomonas (depolarization) reinhardtii

As used herein, a light-responsive opsin (such as NpHR, BR, AR, GtR3,Mac, ChR2, VChR1, DChR, and ChETA) includes naturally occurring proteinand functional variants, fragments, fusion proteins comprising thefragments, or the full length protein. For example, the signal peptidemay be deleted. A variant may have an amino acid sequence at least aboutany of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to the naturally occurring protein sequence. A functionalvariant may have the same or similar hyperpolarization function ordepolarization function as the naturally occurring protein.

Enhanced Intracellular Transport Amino Acid Motifs

The present disclosure provides for the modification of light-responsiveopsin proteins expressed in a cell by the addition of one or more aminoacid sequence motifs which enhance transport to the plasma membranes ofmammalian cells. Light-responsive opsin proteins having componentsderived from evolutionarily simpler organisms may not be expressed ortolerated by mammalian cells or may exhibit impaired subcellularlocalization when expressed at high levels in mammalian cells.Consequently, in some embodiments, the light-responsive opsin proteinsexpressed in a cell can be fused to one or more amino acid sequencemotifs selected from the group consisting of a signal peptide, anendoplasmic reticulum (ER) export signal, a membrane trafficking signal,and/or an N-terminal golgi export signal. The one or more amino acidsequence motifs which enhance light-responsive opsin protein transportto the plasma membranes of mammalian cells can be fused to theN-terminus, the C-terminus, or to both the N- and C-terminal ends of thelight-responsive opsin protein. Optionally, the light-responsive opsinprotein and the one or more amino acid sequence motifs may be separatedby a linker. In some embodiments, the light-responsive opsin protein canbe modified by the addition of a trafficking signal (ts) which enhancestransport of the protein to the cell plasma membrane. In someembodiments, the trafficking signal can be derived from the amino acidsequence of the human inward rectifier potassium channel Kir2.1. Inother embodiments, the trafficking signal can comprise the amino acidsequence KSRITSEGEYIPLDQIDINV.

Additional protein motifs which can enhance light-responsive opsinprotein transport to the plasma membrane of a cell are described in U.S.Patent Application Publication No. 2009/0093403, which is incorporatedherein by reference in its entirety. In some embodiments, the signalpeptide sequence in the protein can be deleted or substituted with asignal peptide sequence from a different protein.

Light-Responsive Chloride Pumps

In some aspects, the light-responsive opsin proteins described hereinare light-responsive chloride pumps. In some aspects of the methodsprovided herein, one or more members of the Halorhodopsin family oflight-responsive chloride pumps are expressed on the plasma membranes ofneurons of the central and peripheral nervous systems.

In some aspects, said one or more light-responsive chloride pumpproteins expressed on the plasma membranes of nerve cells of the centralor peripheral nervous systems can be derived from Natronomonaspharaonis. In some embodiments, the light-responsive chloride pumpproteins can be responsive to amber light as well as red light and canmediate a hyperpolarizing current in the interneuron when thelight-responsive chloride pump proteins are illuminated with amber orred light. The wavelength of light which can activate thelight-responsive chloride pumps can be between about 580 and about 630nm. In some embodiments, the light can be at a wavelength of about 590nm or the light can have a wavelength greater than about 630 nm (e.g.less than about 740 nm). In another embodiment, the light has awavelength of around 630 nm. In some embodiments, the light-responsivechloride pump protein can hyperpolarize a neural membrane for at leastabout 90 minutes when exposed to a continuous pulse of light. In someembodiments, the light-responsive chloride pump protein can comprise anamino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO: 1.Additionally, the light-responsive chloride pump protein can comprisesubstitutions, deletions, and/or insertions introduced into a nativeamino acid sequence to increase or decrease sensitivity to light,increase or decrease sensitivity to particular wavelengths of light,and/or increase or decrease the ability of the light-responsive proteinto regulate the polarization state of the plasma membrane of the cell.In some embodiments, the light-responsive chloride pump protein containsone or more conservative amino acid substitutions. In some embodiments,the light-responsive protein contains one or more non-conservative aminoacid substitutions. The light-responsive protein comprisingsubstitutions, deletions, and/or insertions introduced into the nativeamino acid sequence suitably retains the ability to hyperpolarize theplasma membrane of a neuronal cell in response to light.

Additionally, in other aspects, the light-responsive chloride pumpprotein can comprise a core amino acid sequence at least about 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to thesequence shown in SEQ ID NO: 1 and an endoplasmic reticulum (ER) exportsignal. This ER export signal can be fused to the C-terminus of the coreamino acid sequence or can be fused to the N-terminus of the core aminoacid sequence. In some embodiments, the ER export signal is linked tothe core amino acid sequence by a linker. The linker can comprise any ofabout 5, 10, 20, 30, 40, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275,300, 400, or 500 amino acids in length. The linker may further comprisea fluorescent protein, for example, but not limited to, a yellowfluorescent protein, a red fluorescent protein, a green fluorescentprotein, or a cyan fluorescent protein. In some embodiments, the ERexport signal can comprise the amino acid sequence FXYENE, where X canbe any amino acid. In another embodiment, the ER export signal cancomprise the amino acid sequence VXXSL, where X can be any amino acid.In some embodiments, the ER export signal can comprise the amino acidsequence FCYENEV.

In other aspects, the light-responsive chloride pump proteins providedherein can comprise a light-responsive protein expressed on the cellmembrane, wherein the protein comprises a core amino acid sequence atleast about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to the sequence shown in SEQ ID NO: 1 and a trafficking signal(e.g., which can enhance transport of the light-responsive chloride pumpprotein to the plasma membrane). The trafficking signal may be fused tothe C-terminus of the core amino acid sequence or may be fused to theN-terminus of the core amino acid sequence. In some embodiments, thetrafficking signal can be linked to the core amino acid sequence by alinker which can comprise any of about 5, 10, 20, 30, 40, 50, 75, 100,125, 150, 175, 200, 225, 250, 275, 300, 400, or 500 amino acids inlength. The linker may further comprise a fluorescent protein, forexample, but not limited to, a yellow fluorescent protein, a redfluorescent protein, a green fluorescent protein, or a cyan fluorescentprotein. In some embodiments, the trafficking signal can be derived fromthe amino acid sequence of the human inward rectifier potassium channelKir2.1. In other embodiments, the trafficking signal can comprise theamino acid sequence KSRITSEGEYIPLDQIDINV.

In some aspects, the light-responsive chloride pump protein can comprisea core amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ IDNO: 1 and at least one (such as one, two, three, or more) amino acidsequence motifs which enhance transport to the plasma membranes ofmammalian cells selected from the group consisting of an ER exportsignal, a signal peptide, and a membrane trafficking signal. In someembodiments, the light-responsive chloride pump protein comprises anN-terminal signal peptide, a C-terminal ER Export signal, and aC-terminal trafficking signal. In some embodiments, the C-terminal ERExport signal and the C-terminal trafficking signal can be linked by alinker. The linker can comprise any of about 5, 10, 20, 30, 40, 50, 75,100, 125, 150, 175, 200, 225, 250, 275, 300, 400, or 500 amino acids inlength. The linker can also further comprise a fluorescent protein, forexample, but not limited to, a yellow fluorescent protein, a redfluorescent protein, a green fluorescent protein, or a cyan fluorescentprotein. In some embodiments the ER Export signal can be moreC-terminally located than the trafficking signal. In other embodimentsthe trafficking signal is more C-terminally located than the ER Exportsignal. In some embodiments, the signal peptide comprises the amino acidsequence MTETLPPVTESAVALQAE. In another embodiment, the light-responsivechloride pump protein comprises an amino acid sequence at least 95%identical to SEQ ID NO:2.

Moreover, in other aspects, the light-responsive chloride pump proteinscan comprise a core amino acid sequence at least about 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequenceshown in SEQ ID NO: 1, wherein the N-terminal signal peptide of SEQ IDNO:1 is deleted or substituted. In some embodiments, other signalpeptides (such as signal peptides from other opsins) can be used. Thelight-responsive protein can further comprise an ER transport signaland/or a membrane trafficking signal described herein. In someembodiments, the light-responsive chloride pump protein comprises anamino acid sequence at least 95% identical to SEQ ID NO:3.

In some embodiments, the light-responsive opsin protein is a NpHR opsinprotein comprising an amino acid sequence at least 95%, at least 96%, atleast 97%, at least 98%, at least 99% or 100% identical to the sequenceshown in SEQ ID NO:1. In some embodiments, the NpHR opsin proteinfurther comprises an endoplasmic reticulum (ER) export signal and/or amembrane trafficking signal. For example, the NpHR opsin proteincomprises an amino acid sequence at least 95% identical to the sequenceshown in SEQ ID NO:1 and an endoplasmic reticulum (ER) export signal. Insome embodiments, the amino acid sequence at least 95% identical to thesequence shown in SEQ ID NO:1 is linked to the ER export signal througha linker. In some embodiments, the ER export signal comprises the aminoacid sequence FXYENE, where X can be any amino acid. In anotherembodiment, the ER export signal comprises the amino acid sequenceVXXSL, where X can be any amino acid. In some embodiments, the ER exportsignal comprises the amino acid sequence FCYENEV. In some embodiments,the NpHR opsin protein comprises an amino acid sequence at least 95%identical to the sequence shown in SEQ ID NO:1, an ER export signal, anda membrane trafficking signal. In other embodiments, the NpHR opsinprotein comprises, from the N-terminus to the C-terminus, the amino acidsequence at least 95% identical to the sequence shown in SEQ ID NO:1,the ER export signal, and the membrane trafficking signal. In otherembodiments, the NpHR opsin protein comprises, from the N-terminus tothe C-terminus, the amino acid sequence at least 95% identical to thesequence shown in SEQ ID NO:1, the membrane trafficking signal, and theER export signal. In some embodiments, the membrane trafficking signalis derived from the amino acid sequence of the human inward rectifierpotassium channel Kir2.1. In some embodiments, the membrane traffickingsignal comprises the amino acid sequence K S R I T S E G E Y I P L D Q ID I N V. In some embodiments, the membrane trafficking signal is linkedto the amino acid sequence at least 95% identical to the sequence shownin SEQ ID NO:1 by a linker. In some embodiments, the membranetrafficking signal is linked to the ER export signal through a linker.The linker may comprise any of 5, 10, 20, 30, 40, 50, 75, 100, 125, 150,175, 200, 225, 250, 275, 300, 400, or 500 amino acids in length. Thelinker may further comprise a fluorescent protein, for example, but notlimited to, a yellow fluorescent protein, a red fluorescent protein, agreen fluorescent protein, or a cyan fluorescent protein. In someembodiments, the light-responsive opsin protein further comprises anN-terminal signal peptide. In some embodiments, the light-responsiveopsin protein comprises the amino acid sequence of SEQ ID NO:2. In someembodiments, the light-responsive opsin protein comprises the amino acidsequence of SEQ ID NO:3.

Also provided herein are polynucleotides encoding any of thelight-responsive chloride ion pump proteins described herein, such as alight-responsive protein comprising a core amino acid sequence at leastabout 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to the sequence shown in SEQ ID NO:1, an ER export signal, anda membrane trafficking signal. In another embodiment, thepolynucleotides comprise a sequence which encodes an amino acid at least95% identical to SEQ ID NO:2 and/or SEQ ID NO:3. The polynucleotides maybe in an expression vector (such as, but not limited to, a viral vectordescribed herein). The polynucleotides may be used for expression of thelight-responsive chloride ion pump proteins in neurons of the central orperipheral nervous systems.

Further disclosure related to light-responsive chloride pump proteinscan be found in U.S. Patent Application Publication Nos: 2009/0093403and 2010/0145418 as well as in International Patent Application No:PCT/US2011/028893, the disclosures of each of which are herebyincorporated by reference in their entireties.

Light-Responsive Proton Pumps

In some aspects, the light-responsive opsin proteins described hereinare light-responsive proton pumps. In some aspects of the compositionsand methods provided herein, one or more light-responsive proton pumpsare expressed on the plasma membranes of neurons of the central orperipheral nervous systems.

In some embodiments, the light-responsive proton pump protein can beresponsive to blue light and can be derived from Guillardia theta,wherein the proton pump protein can be capable of mediating ahyperpolarizing current in the cell when the cell is illuminated withblue light. The light can have a wavelength between about 450 and about495 nm or can have a wavelength of about 490 nm. In another embodiment,the light-responsive proton pump protein can comprise an amino acidsequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% identical to the sequence shown in SEQ ID NO:4. Thelight-responsive proton pump protein can additionally comprisesubstitutions, deletions, and/or insertions introduced into a nativeamino acid sequence to increase or decrease sensitivity to light,increase or decrease sensitivity to particular wavelengths of light,and/or increase or decrease the ability of the light-responsive protonpump protein to regulate the polarization state of the plasma membraneof the cell. Additionally, the light-responsive proton pump protein cancontain one or more conservative amino acid substitutions and/or one ormore non-conservative amino acid substitutions. The light-responsiveproton pump protein comprising substitutions, deletions, and/orinsertions introduced into the native amino acid sequence suitablyretains the ability to hyperpolarize the plasma membrane of a neuronalcell in response to light.

In other aspects of the methods disclosed herein, the light-responsiveproton pump protein can comprise a core amino acid sequence at leastabout 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to the sequence shown in SEQ ID NO:4 and at least one (such asone, two, three, or more) amino acid sequence motifs which enhancetransport to the plasma membranes of mammalian cells selected from thegroup consisting of a signal peptide, an ER export signal, and amembrane trafficking signal. In some embodiments, the light-responsiveproton pump protein comprises an N-terminal signal peptide and aC-terminal ER export signal. In some embodiments, the light-responsiveproton pump protein comprises an N-terminal signal peptide and aC-terminal trafficking signal. In some embodiments, the light-responsiveproton pump protein comprises an N-terminal signal peptide, a C-terminalER Export signal, and a C-terminal trafficking signal. In someembodiments, the light-responsive proton pump protein comprises aC-terminal ER Export signal and a C-terminal trafficking signal. In someembodiments, the C-terminal ER Export signal and the C-terminaltrafficking signal are linked by a linker. The linker can comprise anyof about 5, 10, 20, 30, 40, 50, 75, 100, 125, 150, 175, 200, 225, 250,275, 300, 400, or 500 amino acids in length. The linker may furthercomprise a fluorescent protein, for example, but not limited to, ayellow fluorescent protein, a red fluorescent protein, a greenfluorescent protein, or a cyan fluorescent protein. In some embodimentsthe ER Export signal is more C-terminally located than the traffickingsignal. In some embodiments the trafficking signal is more C-terminallylocated than the ER Export signal.

Also provided herein are isolated polynucleotides encoding any of thelight-responsive proton pump proteins described herein, such as alight-responsive proton pump protein comprising a core amino acidsequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% identical to the sequence shown in SEQ ID NO:4. Alsoprovided herein are expression vectors (such as a viral vector describedherein) comprising a polynucleotide encoding the proteins describedherein, such as a light-responsive proton pump protein comprising a coreamino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO:4.The polynucleotides may be used for expression of the light-responsiveproton pumps in neural cells of the central or peripheral nervoussystems.

Further disclosure related to light-responsive proton pump proteins canbe found in International Patent Application No. PCT/US2011/028893, thedisclosure of which is hereby incorporated by reference in its entirety.

Light-Activated Cation Channel Proteins

In some aspects, the light-responsive opsin proteins described hereinare light-activated cation channel proteins. In some aspects of themethods provided herein, one or more light-activated cation channels canbe expressed on the plasma membranes of the neural cells of the centralor peripheral nervous systems.

In some aspects, the light-activated cation channel protein can bederived from Chlamydomonas reinhardtii, wherein the cation channelprotein can be capable of mediating a depolarizing current in the cellwhen the cell is illuminated with light. In another embodiment, thelight-activated cation channel protein can comprise an amino acidsequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% identical to the sequence shown in SEQ ID NO:5. The lightused to activate the light-activated cation channel protein derived fromChlamydomonas reinhardtii can have a wavelength between about 460 andabout 495 nm or can have a wavelength of about 480 nm. Additionally, thelight can have an intensity of at least about 100 Hz. In someembodiments, activation of the light-activated cation channel derivedfrom Chlamydomonas reinhardtii with light having an intensity of 100 Hzcan cause depolarization-induced synaptic depletion of the neuronsexpressing the light-activated cation channel. The light-activatedcation channel protein can additionally comprise substitutions,deletions, and/or insertions introduced into a native amino acidsequence to increase or decrease sensitivity to light, increase ordecrease sensitivity to particular wavelengths of light, and/or increaseor decrease the ability of the light-activated cation channel protein toregulate the polarization state of the plasma membrane of the cell.

Additionally, the light-activated cation channel protein can contain oneor more conservative amino acid substitutions and/or one or morenon-conservative amino acid substitutions. The light-activated protonpump protein comprising substitutions, deletions, and/or insertionsintroduced into the native amino acid sequence suitably retains theability to depolarize the plasma membrane of a neuronal cell in responseto light.

Further disclosure related to light-activated cation channel proteinscan be found in U.S. Patent Application Publication No. 2007/0054319 andInternational Patent Application Publication Nos. WO 2009/131837 and WO2007/024391, the disclosures of each of which are hereby incorporated byreference in their entireties.

Step Function Opsins and Stabilized Step Function Opsins

In other embodiments, the light-activated cation channel protein can bea step function opsin (SFO) protein or a stabilized step function opsin(SSFO) protein that can have specific amino acid substitutions at keypositions throughout the retinal binding pocket of the protein. In someembodiments, the SFO protein can have a mutation at amino acid residueC128 of SEQ ID NO:5. In other embodiments, the SFO protein has a C128Amutation in SEQ ID NO:5. In other embodiments, the SFO protein has aC128S mutation in SEQ ID NO:5. In another embodiment, the SFO proteinhas a C128T mutation in SEQ ID NO:5. In some embodiments, the SFOprotein can comprise an amino acid sequence at least about 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to thesequence shown in SEQ ID NO:6.

In some embodiments, the SSFO protein can have a mutation at amino acidresidue D156 of SEQ ID NO:5. In other embodiments, the SSFO protein canhave a mutation at both amino acid residues C128 and D156 of SEQ IDNO:5. In one embodiment, the SSFO protein has an C128S and a D156Amutation in SEQ ID NO:5. In another embodiment, the SSFO protein cancomprise an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQID NO:7.

In some embodiments the SFO or SSFO proteins provided herein can becapable of mediating a depolarizing current in the cell when the cell isilluminated with blue light. In other embodiments, the light can have awavelength of about 445 nm. Additionally, the light can have anintensity of about 100 Hz. In some embodiments, activation of the SFO orSSFO protein with light having an intensity of 100 Hz can causedepolarization-induced synaptic depletion of the neurons expressing theSFO or SSFO protein. In some embodiments, each of the disclosed stepfunction opsin and stabilized step function opsin proteins can havespecific properties and characteristics for use in depolarizing themembrane of a neuronal cell in response to light.

Further disclosure related to SFO or SSFO proteins can be found inInternational Patent Application Publication No. WO 2010/056970 and U.S.Provisional Patent Application Nos. 61/410,704 and 61/511,905, thedisclosures of each of which are hereby incorporated by reference intheir entireties.

C1V1 Chimeric Cation Channels

In other embodiments, the light-activated cation channel protein can bea C1V1 chimeric protein derived from the VChR1 protein of Volvox carteriand the ChR1 protein from Chlamydomonas reinhardti, wherein the proteincomprises the amino acid sequence of VChR1 having at least the first andsecond transmembrane helices replaced by the first and secondtransmembrane helices of ChR1; is responsive to light; and is capable ofmediating a depolarizing current in the cell when the cell isilluminated with light. In some embodiments, the C1V1 protein canfurther comprise a replacement within the intracellular loop domainlocated between the second and third transmembrane helices of thechimeric light responsive protein, wherein at least a portion of theintracellular loop domain is replaced by the corresponding portion fromChR1. In another embodiment, the portion of the intracellular loopdomain of the C1V1 chimeric protein can be replaced with thecorresponding portion from ChR1 extending to amino acid residue A145 ofthe ChR1. In other embodiments, the C1V1 chimeric protein can furthercomprise a replacement within the third transmembrane helix of thechimeric light responsive protein, wherein at least a portion of thethird transmembrane helix is replaced by the corresponding sequence ofChR1. In yet another embodiment, the portion of the intracellular loopdomain of the C1V1 chimeric protein can be replaced with thecorresponding portion from ChR1 extending to amino acid residue W163 ofthe ChR1. In other embodiments, the C1V1 chimeric protein can comprisean amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO:8.

In some embodiments, the C1V1 protein can mediate a depolarizing currentin the cell when the cell is illuminated with green light. In otherembodiments, the light can have a wavelength of between about 540 nm toabout 560 nm. In some embodiments, the light can have a wavelength ofabout 542 nm. In some embodiments, the C1V1 chimeric protein is notcapable of mediating a depolarizing current in the cell when the cell isilluminated with violet light. In some embodiments, the chimeric proteinis not capable of mediating a depolarizing current in the cell when thecell is illuminated with light having a wavelength of about 405 nm.Additionally, the light can have an intensity of about 100 Hz. In someembodiments, activation of the C1V1 chimeric protein with light havingan intensity of 100 Hz can cause depolarization-induced synapticdepletion of the neurons expressing the C1V1 chimeric protein. In someembodiments, the disclosed C1V1 chimeric protein can have specificproperties and characteristics for use in depolarizing the membrane of aneuronal cell in response to light.

C1V1 Chimeric Mutant Variants

In some aspects, the invention can include polypeptides comprisingsubstituted or mutated amino acid sequences, wherein the mutantpolypeptide retains the characteristic light-responsive nature of theprecursor C1V1 chimeric polypeptide but may also possess alteredproperties in some specific aspects. For example, the mutantlight-activated C1V1 chimeric proteins described herein can exhibit anincreased level of expression both within an animal cell or on theanimal cell plasma membrane; an altered responsiveness when exposed todifferent wavelengths of light, particularly red light; and/or acombination of traits whereby the chimeric C1V1 polypeptide possess theproperties of low desensitization, fast deactivation, low violet-lightactivation for minimal cross-activation with other light-activatedcation channels, and/or strong expression in animal cells.

Accordingly, provided herein are C1V1 chimeric light-activated proteinsthat can have specific amino acid substitutions at key positionsthroughout the retinal binding pocket of the VChR1 portion of thechimeric polypeptide. In some embodiments, the C1V1 protein can have amutation at amino acid residue E122 of SEQ ID NO:7. In some embodiments,the C1V1 protein can have a mutation at amino acid residue E162 of SEQID NO:7. In other embodiments, the C1V1 protein can have a mutation atboth amino acid residues E162 and E122 of SEQ ID NO:7. In otherembodiments, the C1V1 protein can comprise an amino acid sequence atleast about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to the sequence shown in SEQ ID NO:9, SEQ ID NO:10, or SEQ IDNO:11. In some embodiments, each of the disclosed mutant C1V1 chimericproteins can have specific properties and characteristics for use indepolarizing the membrane of an animal cell in response to light.

In some aspects, the C1V1-E122 mutant chimeric protein is capable ofmediating a depolarizing current in the cell when the cell isilluminated with light. In some embodiments the light can be greenlight. In other embodiments, the light can have a wavelength of betweenabout 540 nm to about 560 nm. In some embodiments, the light can have awavelength of about 546 nm. In other embodiments, the C1V1-E122 mutantchimeric protein can mediate a depolarizing current in the cell when thecell is illuminated with red light. In some embodiments, the red lightcan have a wavelength of about 630 nm. In some embodiments, theC1V1-E122 mutant chimeric protein does not mediate a depolarizingcurrent in the cell when the cell is illuminated with violet light. Insome embodiments, the chimeric protein does not mediate a depolarizingcurrent in the cell when the cell is illuminated with light having awavelength of about 405 nm. Additionally, the light can have anintensity of about 100 Hz. In some embodiments, activation of theC1V1-E122 mutant chimeric protein with light having an intensity of 100Hz can cause depolarization-induced synaptic depletion of the neuronsexpressing the C1V1-E122 mutant chimeric protein. In some embodiments,the disclosed C1V1-E122 mutant chimeric protein can have specificproperties and characteristics for use in depolarizing the membrane of aneuronal cell in response to light.

In other aspects, the C1V1-E162 mutant chimeric protein is capable ofmediating a depolarizing current in the cell when the cell isilluminated with light. In some embodiments the light can be greenlight. In other embodiments, the light can have a wavelength of betweenabout 540 nm to about 535 nm. In some embodiments, the light can have awavelength of about 542 nm. In other embodiments, the light can have awavelength of about 530 nm. In some embodiments, the C1V1-E162 mutantchimeric protein does not mediate a depolarizing current in the cellwhen the cell is illuminated with violet light. In some embodiments, thechimeric protein does not mediate a depolarizing current in the cellwhen the cell is illuminated with light having a wavelength of about 405nm. Additionally, the light can have an intensity of about 100 Hz. Insome embodiments, activation of the C1V1-E162 mutant chimeric proteinwith light having an intensity of 100 Hz can causedepolarization-induced synaptic depletion of the neurons expressing theC1V1-E162 mutant chimeric protein. In some embodiments, the disclosedC1V1-E162 mutant chimeric protein can have specific properties andcharacteristics for use in depolarizing the membrane of a neuronal cellin response to light.

In yet other aspects, the C1V1-E122/E162 mutant chimeric protein iscapable of mediating a depolarizing current in the cell when the cell isilluminated with light. In some embodiments the light can be greenlight. In other embodiments, the light can have a wavelength of betweenabout 540 nm to about 560 nm. In some embodiments, the light can have awavelength of about 546 nm. In some embodiments, the C1V1-E122/E162mutant chimeric protein does not mediate a depolarizing current in thecell when the cell is illuminated with violet light. In someembodiments, the chimeric protein does not mediate a depolarizingcurrent in the cell when the cell is illuminated with light having awavelength of about 405 nm. In some embodiments, the C1V1-E122/E162mutant chimeric protein can exhibit less activation when exposed toviolet light relative to C1V1 chimeric proteins lacking mutations atE122/E162 or relative to other light-activated cation channel proteins.Additionally, the light can have an intensity of about 100 Hz. In someembodiments, activation of the C1V1-E122/E162 mutant chimeric proteinwith light having an intensity of 100 Hz can causedepolarization-induced synaptic depletion of the neurons expressing theC1V1-E122/E162 mutant chimeric protein. In some embodiments, thedisclosed C1V1-E122/E162 mutant chimeric protein can have specificproperties and characteristics for use in depolarizing the membrane of aneuronal cell in response to light.

Further disclosure related to C1V1 chimeric cation channels as well asmutant variants of the same can be found in U.S. Provisional PatentApplication Nos. 61/410,736, 61/410,744, and 61/511,912, the disclosuresof each of which are hereby incorporated by reference in theirentireties.

Polynucleotides

The disclosure also provides polynucleotides comprising a nucleotidesequence encoding a light-responsive opsin protein described herein. Insome embodiments, the polynucleotide comprises an expression cassette.In some embodiments, the polynucleotide is a vector comprising theabove-described nucleic acid(s). In some embodiments, the nucleic acidencoding a light-activated protein of the disclosure is operably linkedto a promoter. Promoters are well known in the art. Any promoter thatfunctions in the host cell can be used for expression of thelight-responsive opsin proteins and/or any variant thereof of thepresent disclosure. In one embodiment, the promoter used to driveexpression of the light-responsive opsin proteins is a promoter that isspecific to motor neurons. In another embodiment, the promoter used todrive expression of the light-responsive opsin proteins is a promoterthat is specific to central nervous system neurons. In otherembodiments, the promoter is capable of driving expression of thelight-responsive opsin proteins in neurons of both the sympatheticand/or the parasympathetic nervous systems. Initiation control regionsor promoters, which are useful to drive expression of thelight-responsive opsin proteins or variant thereof in a specific animalcell are numerous and familiar to those skilled in the art. Virtuallyany promoter capable of driving these nucleic acids can be used.Examples of motor neuron-specific genes can be found, for example, inKudo, et al., Human Mol. Genetics, 2010, 19(16): 3233-3253, the contentsof which are hereby incorporated by reference in their entirety. In someembodiments, the promoter used to drive expression of thelight-activated protein can be the Thy1 promoter, which is capable ofdriving robust expression of transgenes in neurons of both the centraland peripheral nervous systems (See, e.g., Llewellyn, et al., 2010, Nat.Med., 16(10):1161-1166). In other embodiments, the promoter used todrive expression of the light-responsive opsin protein can be the EF1αpromoter, a cytomegalovirus (CMV) promoter, the CAG promoter, thesinapsin promoter, or any other ubiquitous promoter capable of drivingexpression of the light-responsive opsin proteins in the peripheraland/or central nervous system neurons of mammals.

Also provided herein are vectors comprising a nucleotide sequenceencoding a light-responsive opsin protein or any variant thereofdescribed herein. The vectors that can be administered according to thepresent invention also include vectors comprising a nucleotide sequencewhich encodes an RNA (e.g., an mRNA) that when transcribed from thepolynucleotides of the vector will result in the accumulation oflight-responsive opsin proteins on the plasma membranes of target animalcells. Vectors which may be used, include, without limitation,lentiviral, HSV, adenoviral, and andeno-associated viral (AAV) vectors.Lentiviruses include, but are not limited to HIV-1, HIV-2, SW, FW andEIAV. Lentiviruses may be pseudotyped with the envelope proteins ofother viruses, including, but not limited to VSV, rabies, Mo-MLV,baculovirus and Ebola. Such vectors may be prepared using standardmethods in the art.

In some embodiments, the vector is a recombinant AAV vector. AAV vectorsare DNA viruses of relatively small size that can integrate, in a stableand site-specific manner, into the genome of the cells that they infect.They are able to infect a wide spectrum of cells without inducing anyeffects on cellular growth, morphology or differentiation, and they donot appear to be involved in human pathologies. The AAV genome has beencloned, sequenced and characterized. It encompasses approximately 4700bases and contains an inverted terminal repeat (ITR) region ofapproximately 145 bases at each end, which serves as an origin ofreplication for the virus. The remainder of the genome is divided intotwo essential regions that carry the encapsidation functions: theleft-hand part of the genome, that contains the rep gene involved inviral replication and expression of the viral genes; and the right-handpart of the genome, that contains the cap gene encoding the capsidproteins of the virus.

AAV vectors may be prepared using standard methods in the art.Adeno-associated viruses of any serotype are suitable (See, e.g.,Blacklow, pp. 165-174 of “Parvoviruses and Human Disease” J. R.Pattison, ed. (1988); Rose, Comprehensive Virology 3:1, 1974; P.Tattersall “The Evolution of Parvovirus Taxonomy” in Parvoviruses (J RKerr, S F Cotmore. M E Bloom, R M Linden, C R Parrish, Eds.) p 5-14,Hudder Arnold, London, UK (2006); and D E Bowles, J E Rabinowitz, R JSamulski “The Genus Dependovirus” (J R Kerr, S F Cotmore. M E Bloom, R MLinden, C R Parrish, Eds.) p 15-23, Hudder Arnold, London, UK (2006),the disclosures of each of which are hereby incorporated by referenceherein in their entireties). Methods for purifying for vectors may befound in, for example, U.S. Pat. Nos. 6,566,118, 6,989,264, and6,995,006 and International Patent Application Publication No.:WO/1999/011764 titled “Methods for Generating High Titer Helper-freePreparation of Recombinant AAV Vectors”, the disclosures of which areherein incorporated by reference in their entirety. Preparation ofhybrid vectors is described in, for example, PCT Application No.PCT/US2005/027091, the disclosure of which is herein incorporated byreference in its entirety. The use of vectors derived from the AAVs fortransferring genes in vitro and in vivo has been described (See e.g.,International Patent Application Publication Nos.: WO 91/18088 and WO93/09239; U.S. Pat. Nos. 4,797,368, 6,596,535, and 5,139,941; andEuropean Patent No.: 0488528, all of which are hereby incorporated byreference herein in their entireties). These publications describevarious AAV-derived constructs in which the rep and/or cap genes aredeleted and replaced by a gene of interest, and the use of theseconstructs for transferring the gene of interest in vitro (into culturedcells) or in vivo (directly into an organism). The replication defectiverecombinant AAVs according to the invention can be prepared byco-transfecting a plasmid containing the nucleic acid sequence ofinterest flanked by two AAV inverted terminal repeat (UR) regions, and aplasmid carrying the AAV encapsidation genes (rep and cap genes), into acell line that is infected with a human helper virus (for example, anadenovirus). The AAV recombinants that are produced are then purified bystandard techniques.

In some embodiments, the vector(s) for use in the methods of theinvention are encapsidated into a virus particle (e.g. AAV virusparticle including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5,AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, andAAV16). Accordingly, the invention includes a recombinant virus particle(recombinant because it contains a recombinant polynucleotide)comprising any of the vectors described herein. Methods of producingsuch particles are known in the art and are described in U.S. Pat. No.6,596,535, the disclosure of which is hereby incorporated by referencein its entirety.

Delivery of Light-Responsive Opsin Proteins and Lanthanide-DopedNanoparticles

In some aspects, polynucleotides encoding the light-responsive opsinproteins disclosed herein (for example, an AAV1 vector) can be delivereddirectly to neurons of the central or peripheral nervous system with aneedle, catheter, or related device, using neurosurgical techniquesknown in the art, such as by stereotactic injection (See, e.g., Stein etal., J. Virol., 1999, 73:34243429; Davidson et al., Proc. Nat. Acad.Sci. U.S.A., 2000, 97:3428-3432; Davidson et al., Nat. Genet., 1993,3:219-223; and Alisky & Davidson, Hum. Gene Ther., 2000, 11:2315-2329,the contents of each of which are hereby incorporated by referenceherein in their entireties) or fluoroscopy. In some embodiments, thepolynucleotide encoding the light-responsive opsin proteins disclosedherein (for example, an AAV1 vector) can be delivered to neurons of theperipheral nervous system by injection into any one of the spinal nerves(such as the cervical spinal nerves, the thoracic spinal nerves, thelumbar spinal nerves, the sacral spinal nerves, and/or the coccygealspinal nerves).

Other methods to deliver the light-responsive opsin proteins to thenerves of interest can also be used, such as, but not limited to,transfection with ionic lipids or polymers, electroporation, opticaltransfection, impalefection, or via gene gun.

In another aspect, the polynucleotide encoding the light-responsiveopsin proteins disclosed herein (for example, an AAV2 vector) can bedelivered directly to muscles innervated by the neurons of theperipheral nervous system. Because of the limitations inherent ininjecting viral vectors directly into the specific cell bodies whichinnvervate particular muscles, researchers have attempted to delivertransgenes to peripheral neurons by injecting viral vectors directlyinto muscle. These experiments have shown that some viral serotypes suchas adenovirus, AAV2, and Rabies glycoprotein-pseudotyped lentivirus canbe taken up by muscle cells and retrogradely transported to motorneurons across the neuromuscular synapse (See, e.g., Azzouz et al.,2009, Antioxid Redox Signal., 11(7):1523-34; Kaspar et al., 2003,Science, 301(5634):839-842; Manabe et al., 2002, Apoptosis,7(4):329-334, the disclosures of each of which are herein incorporatedby reference in their entireties).

Accordingly, in some embodiments, the vectors expressing thelight-responsive opsin proteins disclosed herein (for example, an AAV2vector) can be delivered to the neurons responsible for the innervationof muscles by direct injection into the muscle of interest.

The lanthanide-doped nanoparticles disclosed herein can be delivered toneurons expressing one or more light-responsive opsin proteins by anyroute, such as intravascularly, intracranially, intracerebrally,intramuscularly, intradermally, intravenously, intraocularly, orally,nasally, topically, or by open surgical procedure, depending upon theanatomical site or sites to which the nanoparticles are to be delivered.The nanoparticles can additionally be delivered by the same route usedfor delivery of the polynucleotide vectors expressing thelight-responsive opsin proteins, such as any of those described above.The nanoparticles can also be administered in an open manner, as in theheart during open heart surgery, or in the brain during stereotacticsurgery, or by intravascular interventional methods using cathetersgoing to the blood supply of specific organs, or by other interventionalmethods.

Pharmaceutical compositions used for the delivery and/or storage ofpolynucleotides encoding the light-responsive opsin proteins disclosedherein and/or the lanthanide-doped nanoparticles disclosed herein can beformulated according to known methods for preparing pharmaceuticallyuseful compositions. Formulations are described in a number of sourceswhich are well known and readily available to those skilled in the art.For example, Remington's Pharmaceutical Sciences (Martin E W, 1995,Easton Pa., Mack Publishing Company, 19^(th) ed.) describes formulationswhich can be used in connection with the subject invention. Formulationssuitable for parenteral administration include, for example, aqueoussterile injection solutions, which may contain antioxidants, buffers,bacteriostats, and solutes which render the formulation isotonic withthe blood of the intended recipient; and aqueous and non-aqueous sterilesuspensions which may include suspending agents and thickening agents.The formulations may be presented in unit-dose or multi-dose containers,for example, sealed ampoules and vials, and may be stored in a freezedried (lyophilized) condition requiring only the condition of thesterile liquid carrier, for example, water for injections, prior to use.

The lanthanide-doped nanoparticles may also be administeredintravenously or intraperitoneally by infusion or injection. Solutionsof the nanoparticles and/or cells can be prepared in water, optionallymixed with a nontoxic surfactant. Dispersions can also be prepared inglycerol, liquid polyethylene glycols, triacetin, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The pharmaceutical dosage forms suitable for injection or infusion ofthe lanthanide-doped nanoparticles described herein can include sterileaqueous solutions or dispersions or sterile powders comprising theactive ingredient which are adapted for the extemporaneous preparationof sterile injectable or infusible solutions or dispersions. The liquidcarrier or vehicle can be a solvent or liquid dispersion mediumcomprising, for example, water, ethanol, a polyol (for example,glycerol, propylene glycol, liquid polyethylene glycols, and the like),vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof.The prevention of the action of microorganisms can be brought about byvarious antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like.

Sources of Infrared or Near Infrared Electromagnetic Radiation

Any device that is capable of producing a source of electromagneticradiation having a wavelength in the infrared (IR) or near infrared(NIR) spectrum may be used to activate one or more light-responsiveproteins expressed on the surface of a neuron in combination with thelanthanide-doped nanoparticles described herein. The IR or NIR sourcecan be configured to provide optical stimulus to a specific targetregion of the brain. The IR or NIR source can additionally providecontinuous IR or NIR electromagnetic radiation and/or pulsed IR or NIRelectromagnetic radiation, and may be programmable to provide IR or NIRelectromagnetic radiation in pre-determined pulse sequences.

In other aspects, the implantable IR or NIR source does not requirephysical tethering to an external power source. In some embodiments, thepower source can be an internal battery for powering the IR or NIRsource. In another embodiment, the implantable IR or NIR source cancomprise an external antenna for receiving wirelessly transmittedelectromagnetic energy from an external power source for powering the IRor NIR source. The wirelessly transmitted electromagnetic energy can bea radio wave, a microwave, or any other electromagnetic energy sourcethat can be transmitted from an external source to power the IR orNIR-generating source. In one embodiment, the IR or NIR source iscontrolled by an integrated circuit produced using semiconductor orother processes known in the art.

In some aspects, the implantable IR or NIR electromagnetic radiationsource can be externally activated by an external controller. Theexternal controller can comprise a power generator which can be mountedto a transmitting coil. In some embodiments of the external controller,a battery can be connected to the power generator, for providing powerthereto. A switch can be connected to the power generator, allowing anindividual to manually activate or deactivate the power generator. Insome embodiments, upon activation of the switch, the power generator canprovide power to the IR or NIR electromagnetic radiation source throughelectromagnetic coupling between the transmitting coil on the externalcontroller and the external antenna of the implantable IR or NIR source.When radio-frequency magnetic inductance coupling is used, theoperational frequency of the radio wave can be between about 1 and 20MHz, inclusive, including any values in between these numbers (forexample, about 1 MHz, about 2 MHz, about 3 MHz, about 4 MHz, about 5MHz, about 6 MHz, about 7 MHz, about 8 MHz, about 9 MHz, about 10 MHz,about 11 MHz, about 12 MHz, about 13 MHz, about 14 MHz, about 15 MHz,about 16 MHz, about 17 MHz, about 18 MHz, about 19 MHz, or about 20MHz). However, other coupling techniques may be used, such as an opticalreceiver or a biomedical telemetry system (See, e.g., Kiourti,“Biomedical Telemetry: Communication between Implanted Devices and theExternal World, Opticon1826, (8): Spring, 2010).

In some aspects, the intensity of the IR or NIR electromagneticradiation reaching the neural cells (such as neural cells expressing oneor more light-responsive opsin proteins) produced by the IR or NIRelectromagnetic radiation source has an intensity of any of about 0.05mW/mm², 0.1 mW/mm², 0.2 mW/mm², 0.3 mW/mm², 0.4 mW/mm², 0.5 mW/mm²,about 0.6 mW/mm², about 0.7 mW/mm², about 0.8 mW/mm², about 0.9 mW/mm²,about 1.0 mW/mm², about 1.1 mW/mm², about 1.2 mW/mm², about 1.3 mW/mm²,about 1.4 mW/mm², about 1.5 mW/mm², about 1.6 mW/mm², about 1.7 mW/mm²,about 1.8 mW/mm², about 1.9 mW/mm², about 2.0 mW/mm², about 2.1 mW/mm²,about 2.2 mW/mm², about 2.3 mW/mm², about 2.4 mW/mm², about 2.5 mW/mm²,about 3 mW/mm², about 3.5 mW/mm², about 4 mW/mm², about 4.5 mW/mm²,about 5 mW/mm², about 5.5 mW/mm², about 6 mW/mm², about 7 mW/mm², about8 mW/mm², about 9 mW/mm², or about 10 mW/mm², inclusive, includingvalues in between these numbers.

In other aspects, the IR or NIR electromagnetic radiation produced bythe IR or NIR electromagnetic radiation source can have a wavelengthencompassing the entire infrared spectrum, such as from about 740 nm toabout 300,000 nm. In other embodiments, the IR or NIR electromagneticradiation produced by the IR or NIR electromagnetic radiation source canhave a wavelength corresponding to the NIR spectrum, such as about 740nm to about 1400 nm. In other embodiments, NIR electromagnetic radiationproduced has a wavelength between 700 nm and 1000 nm.

In some aspects, an IR or NIR electromagnetic radiation source is usedto hyperpolarize or depolarize the plasma membranes of neural cells(such as neural cells expressing one or more light-responsive opsinproteins) in the brain or central nervous system of an individual whenused in combination with the lanthanide-doped nanoparticles disclosedherein. In some embodiments, the skull of the individual is surgicallythinned in an area adjacent to the brain region of interest withoutpuncturing the bone. The IR or NIR electromagnetic radiation source canthen be placed directly over the thinned-skull region. In otherembodiments, the IR or NIR electromagnetic radiation generator isimplanted under the skin of the individual directly adjacent to thethinned skull region.

In some aspects, an IR or NIR electromagnetic radiation source is usedto hyperpolarize or depolarize the plasma membranes of neural cells(such as neural cells expressing one or more light-responsive opsinproteins) in the peripheral nervous system of an individual when used incombination with the lanthanide-doped nanoparticles disclosed herein. Insome embodiments, the IR or NIR electromagnetic radiation source issurgically implanted under the skin of the individual directly adjacentto the peripheral neural cell of interest. In other embodiments, the IRor NIR electromagnetic radiation source is placed against the skindirectly adjacent to the peripheral neural cell of interest. In oneembodiment, the IR or NIR electromagnetic radiation source is heldagainst the skin in a bracelet or cuff configuration.

Examples of the IR or NIR electromagnetic radiation sources,particularly those small enough to be implanted under the skin, can befound in U.S. Patent Application Publication Nos.: 2009/0143842,2011/0152969, 2011/0144749, and 2011/0054305, the disclosures of each ofwhich are incorporated by reference herein in their entireties.

In still other aspects, the lanthanide-doped nanoparticles disclosedherein can be exposed to higher wavelength light in the visible spectrum(such as red light) to upconvert the higher wavelength visible lightinto lower wavelength visible light (such as blue or green light). Asdescribed above, light passes through biological tissue poorly. However,when visible light does penetrate into tissues, it typically does so inhigher wavelengths which correspond to red light (for example, betweenabout 620 nm to 740 nm). Accordingly, the lanthanide-doped nanoparticlesdisclosed herein can additionally be used in combination with opticalsources of visible light to upshift wavelengths corresponding to redlight into wavelengths corresponding to green or blue light (forexample, between about 440 nm and 570 nm). Examples of light stimulationdevices, including light sources, can be found in International PatentApplication Nos.: PCT/US08/50628 and PCT/US09/49936 and in Llewellyn etal., 2010, Nat. Med., 16(10):161-165, the disclosures of each of whichare hereby incorporated herein in their entireties.

Methods of the Invention

Depolarization of Neural Cells

Provided herein are methods to depolarize the plasma membrane of aneural cell in an individual comprising placing a plurality oflanthanide-doped nanoparticles in proximity to the neural cell; andexposing the plurality of nanoparticles to electromagnetic radiation inthe infrared (IR) or near infrared (NIR) spectrum, wherein theelectromagnetic radiation in the IR or NIR spectrum is upconverted intolight in the visible spectrum by the nanoparticles, and wherein alight-responsive opsin is expressed on the plasma membrane of the neuralcells and activation of the opsin by the light in the visible spectruminduces the depolarization of the plasma membrane.

Also provided herein is a method to depolarize the plasma membrane of aneural cell in an individual comprising administering a polynucleotideencoding a light-responsive opsin to a neural cell in the brain of anindividual, wherein the light-responsive protein is expressed on theplasma membrane of the neural cell and the opsin is capable of inducingmembrane depolarization of the neural cell when illuminated with lightadministering a plurality of lanthanide-doped nanoparticles in proximityto the neural cell; and exposing the plurality of nanoparticles toelectromagnetic radiation in the infrared (IR) or near (IR) spectrum,wherein the electromagnetic radiation in the IR or near IR spectrum isupconverted into light in the visible spectrum and the activation of theopsin by the light in the visible spectrum induces the depolarization ofthe plasma membrane.

In some embodiments, the light-responsive opsin protein is ChR2, VChR1,or C1V1. In other embodiments, the light-responsive opsin protein isselected from the group consisting of SFO, SSFO, C1V1-E122, C1V1-E162,and C1V1-E122/E162.

The lanthanide metal can be ions or atoms from any of the lanthanideseries of elements, such as Lanthanum, Cerium, Praseodymium, Neodymium,Promethium, Samarium, Europium, Gadolinium, Terbium, Dysprosium,Holmium, Erbium, Thulium, Ytterbium, or Lutetium. In other embodiments,the nanoparticles comprise NaYF4:Yb/X/Gd, wherein X is Er, Tm, or Er/Tm.

The electromagnetic radiation in the IR or near IR spectrum can beupconverted into light having a wavelength of about 450 nm to about 550nm. The light can have wavelengths corresponding to red, yellow, amber,orange, green, or blue light. In some embodiments, the individual is ahuman or a non-human animal. In other embodiments, the neural cell is inthe peripheral nervous system. In another embodiment, the neural cell isin the central nervous system.

Hyperpolarization of Neural Cells

Provided herein are methods to hyperpolarize the plasma membrane of aneural cell in an individual comprising placing a plurality oflanthanide-doped nanoparticles in proximity to the neural cell; andexposing the plurality of nanoparticles to electromagnetic radiation inthe infrared (IR) or near infrared (NIR) spectrum, wherein theelectromagnetic radiation in the IR or NIR spectrum is upconverted intolight in the visible spectrum by the nanoparticles, and wherein alight-responsive opsin is expressed on the plasma membrane of the neuralcells and activation of the opsin by the light in the visible spectruminduces the hyperpolarization of the plasma membrane.

Also provided herein is a method to hyperpolarize the plasma membrane ofa neural cell in an individual comprising administering a polynucleotideencoding a light-responsive opsin to a neural cell in the brain of anindividual, wherein the light-responsive protein is expressed on theplasma membrane of the neural cell and the opsin is capable of inducingmembrane depolarization of the neural cell when illuminated with lightadministering a plurality of lanthanide-doped nanoparticles in proximityto the neural cell; and exposing the plurality of nanoparticles toelectromagnetic radiation in the infrared (IR) or near (IR) spectrum,wherein the electromagnetic radiation in the IR or near IR spectrum isupconverted into light in the visible spectrum and the activation of theopsin by the light in the visible spectrum induces the hyperpolarizationof the plasma membrane.

In some embodiments, the light-responsive opsin protein is an NpHR or aGtR3.

The lanthanide metal can be ions or atoms from any of the lanthanideseries of elements, such as Lanthanum, Cerium, Praseodymium, Neodymium,Promethium, Samarium, Europium, Gadolinium, Terbium, Dysprosium,Holmium, Erbium, Thulium, Ytterbium, or Lutetium. In other embodiments,the nanoparticles comprise NaYF4:Yb/X/Gd, wherein X is Er, Tm, or Er/Tm.

The electromagnetic radiation in the IR or near IR spectrum can beupconverted into light having a wavelength of about 450 nm to about 550nm. The light can have wavelengths corresponding to red, yellow, amber,orange, green, or blue light. In some embodiments, the individual is ahuman or a non-human animal. In other embodiments, the neural cell is inthe peripheral nervous system. In another embodiment, the neural cell isin the central nervous system.

Kits

Also provided herein are kits comprising polynucleotides encoding alight-responsive opsin protein (such as any of the light-responsiveopsin proteins described herein) and lanthanide-doped nanoparticles foruse in any of the methods disclosed herein to alter the membranepolarization state of one or more neurons of the central and/orperipheral nervous system. In some embodiments, the kits furthercomprise an infrared or near infrared electromagnetic radiation source.In other embodiments, the kits further comprise instructions for usingthe polynucleotides and lanthanide-doped nanoparticles described herein.In still other embodiments, the lanthanide-doped nanoparticles describedherein are embedded and/or trapped in a biocompatible material (such asany of the biocompatible materials described above).

Exemplary Embodiments

Aspects of the present disclosure may be more completely understood inconsideration of the detailed description of various embodiments of thepresent disclosure that follows in connection with the accompanyingdrawings. This description and the various embodiments are presented asfollows:

The embodiments and specific applications discussed herein may beimplemented in connection with one or more of the above-describedaspects, embodiments and implementations, as well as with those shown inthe figures and described below. Reference may also be made to Wang etal., 2010, Nature, 463(7284):1061-5, which is fully incorporated hereinby reference. For further details on light responsive molecules and/oropsins, including methodology, devices and substances, reference mayalso be made to the following background publications: U.S. PatentPublication No. 2010/0190229, entitled “System for Optical Stimulationof Target Cells” to Zhang et al.; U.S. Patent Publication No.2007/0261127, entitled “System for Optical Stimulation of Target Cells”to Boyden et al. These applications form part of the provisional patentdocument and are fully-incorporated herein by reference. Consistent withthese publications, numerous opsins can be used in mammalian cells invivo and in vitro to provide optical stimulation and control of targetcells. For example, when ChR2 is introduced into anelectrically-excitable cell, such as a neuron, light activation of theChR2 channelrhodopsin can result in excitation and/or firing of thecell. In instances when NpHR is introduced into anelectrically-excitable cell, such as a neuron, light activation of theNpHR opsin can result in inhibition of firing of the cell. These andother aspects of the disclosures of the above-referenced patentapplications may be useful in implementing various aspects of thepresent disclosure.

In various embodiments of the present disclosure, minimally invasivedelivery of light, for example as can be useful for manipulation ofneural circuits with optogenetics, using near infrared up-conversionnanocrystals, is achieved. This is used to avoid the implantation oflight sources within living tissues, including, for example, a subject'sbrain. Mammalian tissue has a transparency window in near infrared partof the spectrum (700-1000 nm). Accordingly, aspects of the presentdisclosure relate to the use of nanoparticles for the purpose of using(near) infrared light to deliver energy into the depth of a brain byconverting the infrared light into visible wavelengths at a site ofinterest.

In certain embodiments, delivering visible wavelengths at a site ofinterest within the brain is achieved through a process of opticalupconversion in Lanthanide-doped nanocrystals. During upconversion 3-4photons are absorbed by the material which then emits one photon withthe energy ˜1.5-2 times the energy of absorbed photons. For exampleNaYF4:Yb/X/Gd nanocrystals can absorb 980 nm light and emit light withspectra centered between 450-550 nm depending on the nature and relativecontent of dopants (X=Er, Tm, Er/Tm). For more information regardingmodifying the light emitted from the nanoparticles, see Wang et al.,Nature, 2010, 463(7284):1061-5, the disclosure of which is incorporatedby reference herein in its entirety.

In certain embodiments a single step surgery is performed to modify atarget cell population and provide nanoparticles to convert nearinfrared light to visible light that stimulates the modified target cellpopulation. During the surgery, the surgeon injects both anadeno-associated virus carrying an opsin gene and a nanoparticlesolution to a site of interest.

The virus is optimized to only infect the target cell population.Similarly, the nanoparticles are functionalized with antibodies so thatthe nanoparticles anchor to the target cell population as well. Incertain more specific embodiments the target cell population is aparticular neuron type. After surgery is completed, a LED that emitsnear infrared light is placed on a thinned portion of the patient'sskull, underneath the skin. A battery can also be implanted underneaththe skin to power the LED. In certain embodiments the battery hascharacteristics similar to those of a pacemaker battery. Amicrocontroller can be used to control the battery to deliver energy tothe LED at specified intervals, resulting in LED light pulses atspecified intervals.

Certain aspects of the present disclosure are directed to the use ofoptogenetics in vivo. Optogenetics, applied in vivo, relies on lightdelivery to specific neuron populations that can be located deep withinthe brain. Mammalian tissue is highly absorptive and scatters light inthe visible spectrum. However, near infrared light is able to penetrateto deep levels of the brain without excessive absorption or scattering.

Certain aspects of the present disclosure are directed to imbeddingnanoparticles in the brain near target neurons. The nanoparticles can belanthanide doped-nanoparticle. Nanoparticles doped with Lanthanides orwith other dopants can be optimized with respect to a particular opsin'sactivation spectra. As discussed in more detail in Wang et al., Nature,2010, 463(7284):1061-5, the disclosure of which is incorporated byreference herein in its entirety, the spectra of the light emitted fromlanthanide-doped nanocrystals can be manipulated based on which dopantsare used, and how much. Similarly, the light emitted from nanoparticlesdoped with other molecules can be manipulated based on the concentrationof dopants.

The ability to provide different output spectra depending on the dopingof nanoparticles allows for a non-invasive approach to acute neuralmanipulation. A light source, such as a LED can be mounted onto athinned skull under the skin. Depending on the composition ofnanoparticles, and the opsin delivered to the target neurons, aspects ofthe present disclosure can be used for neural excitation or silencing.Similarly, multiple neural populations may be controlled simultaneouslythrough the use of various dopants and opsins in combination.

Turning to FIG. 1, a patient's head 100 is shown. A target (neural) cellpopulation 114 includes light responsive molecules. These lightresponsive molecules can include, but are not necessarily limited to,opsins derived from Channelrhodopsins (e.g. ChR1 or ChR2) orHalorhodopins (NpHR). The specific molecule can be tailored/selectedbased upon the desired effect on the target cell population and/or thewavelength at which the molecules respond to light.

Nanocrystals 110 are introduced near or at the target cell populate.Various embodiments of the present disclosure are directed towardmethods and devices for positioning and maintaining positioning of thenanocrystals near the target cell population. Certain embodiments aredirected toward anchoring the nanocrystals to cells of (or near) thetarget cell population using antibodies.

According to other example embodiments, a structure can be introducedthat includes the nanocrystals. For instance, a mesh structure can becoated with the nanocrystals. The synthetic mesh can be constructed soas to allow the dendrites and axons to pass through the mess withoutallowing the entire neuron (e.g., the cell body) to pass. One example ofsuch a mesh has pores that are on the order of 3-7 microns in diameterand is made from polyethylene terephthalate. This mesh structure can beconstructed with light-responsive cells/neurons contained therein and/orbe placed near the target cell population, which includes thelight-responsive cells. Consistent with another embodiment, one or moretransparent capsules, each containing a solution of nanocrystals, can bepositioned near the target cell populations.

Embodiments of the present disclosure are also directed toward variousoptical sources of stimulation. These sources can include, but are notlimited to, external laser sources and light-emitting diodes (LEDs).Particular aspects of the present disclosure are directed toward therelatively low absorption and/or scattering/diffusion caused byintervening material when the light is at certain wavelengths (e.g.,(near) infrared). Accordingly, the light source can be externallylocated because of the ability to penetrate the tissue with little lossof optical intensity or power. Moreover, reduced diffusion can beparticularly useful for providing a relatively-high spatial-precisionfor the delivery of the light. Thus, embodiments of the presentdisclosure are directed toward multiple target cell populations withrespective nanocrystals that can be individually controlled usingspatially-precise optical stimulus. For instance, the nanocrystals canbe implanted in several locations within the brain. The light source canthen be aimed at a respective and particular location. Multiple lightsources can also be used for simultaneous stimulation of a plurality oflocations.

Consistent with a particular embodiment of the present disclosure, theskull 102 has a thinned portion 106. An LED 104 is located above thethinned portion of the skull and emits near infrared light 108. When theIR hits nanocrystal 110, it is absorbed. The nanocrystal emits visiblelight 112 in response to absorbing the IR light 108. The visible light112 is absorbed by modified cell 114.

The system shown in FIG. 1 allows for delivery of light to a target celldeep within a patient's brain tissue. The light responsive molecule canbe specifically targeted to a neural cell type of interest. Similarly,the nanocrystals 112 are anchored to the neural cell with antibodieschosen based on the type of neural cell 114 being targeted.

Turning to FIG. 2, a group of neurons is illuminated with infrared light208 between 700-1000 nm. Target neurons 214 express an opsin gene,allowing the neurons to be activated or inhibited, depending on whichopsin, and what wavelength of light is absorbed by the neurons 214. Thetarget neurons 214 can be interspersed between other neurons 216. Asshown in inset 202, target neurons 214 are coated with upconvertingnanoparticles 210 that are anchored to the neural membrane viaantibodies. The nanoparticles 210 absorb IR photons and emit visiblephotons that are then absorbed by opsins triggering neural activation.

The system of FIG. 2 can be used with a variety of target neurons 214.The opsin gene 215 expressed in the target neurons 214 is modified basedon the target neuron. Similarly, the antibodies used to anchor thenanoparticles 210 to the target neuron membranes are modified to attachto a specific membrane type. As shown in inset 202, the nanoparticles210 are closely linked to the target neurons so that visible lightphotons emitted by the nanoparticles 210 are absorbed by the targetneurons 214.

FIG. 3 depicts a system that uses multiple light sources, consistentwith an embodiment of the present disclosure. A patient hasnanoparticles located at target locations 308-312. The system includeslight sources 302-306, which can be configured to generate light at afrequency that is upconverted by the nanoparticles located at targetlocations 308-312. Although three light sources are depicted, there canbe any number of light sources. These light sources can be external tothe patient (e.g., a targeting system that directs several light sourcesusing mechanical positioning), using embedded lights sources (e.g., LEDsimplanted on the skull) or combinations thereof. The target locations308-312 include cells that have optically-responsive membrane molecules.These optically-responsive membrane molecules react to light at theupconverted frequency.

Nanoparticles located at the intersection 314 of the light from thedifferent light sources 302-306 receive increased intensity of opticalstimulus relative to other locations, including those locations withinthe path of light from a single light source. In this manner, the lightintensity of each of the light sources can be set below a thresholdlevel. When multiple light sources are directed at the same location,the threshold intensity level can be exceeded at the location. Thisallows for spatial control in three-dimensions and also allows forreduced inadvertent effects on non-targeted tissue. Consistent with oneembodiment, the threshold level can be set according to an amount oflight necessary to cause the desired effect (e.g., excitation orinhibition) on the target cells. Consistent with other embodiments, thethreshold level can be set to avoid adverse effects on non-targetedtissue (e.g., heating).

The use of multiple light sources can also bring about a step-wiseincrease in light inntensity. For instance, a disease model could betested by monitoring the effects of additional stimulation caused by theincrease in light intensity. The use of independent light sources allowsfor relatively simple control over temporal and spatial increases ordecreases. Consistent with other embodiments of the present disclosure,the spatial precision of the light sources can be varied between thedifferent light sources. For example, a first light source can providelight that illuminates the entire target cell location. This allows forall cells within the population to be illuminated. A second light sourcecan provide light having a focal point that illuminates less than all ofthe entire target cell location. The combination of the first and second(or more) light sources can be used to provide different levels ofstimulation within the same cell population.

Embodiments of the present disclosure relate to the use of one or morelight sources operating in a scanning mode. The light source(s) areaimed at specific locations within a target cell population. The effectsof the stimulation can be monitored as the light source is used to scanor otherwise move within the target cell population. This can beparticularly useful in connection with the three-dimensional controlprovided by the use of multiple light sources.

Various embodiments of the present disclosure are directed toward theuse of nanocrystals that emit light at different wavelengths. This canbe particularly useful when using multiple opsins having differentlight-absorption spectrums. The nanocrystals can be targeted towarddifferent opsins and/or placed in the corresponding locations. While thepresent disclosure is amenable to various modifications and alternativeforms, specifics thereof have been shown by way of example in thedrawings and will be described in further detail. It should beunderstood that the intention is not to limit the disclosure to theparticular embodiments and/or applications described. On the contrary,the intention is to cover all modifications, equivalents, andalternatives falling within the spirit and scope of the presentdisclosure.

EXAMPLES Example 1: Use of Lanthanide-Doped Nanoparticles in the Use ofOptogenetics to Hyperpolarize the Cholinergic Interneurons of theNucleus Accumbens

The nucleus accumbens (NAc) is a collection of neurons that forms themain part of the ventral striatum. The NAc is thought to play animportant role in the complex mammalian behaviors associated withreward, pleasure, laughter, addiction, aggression, fear, and the placeboeffect. Cholinergic interneurons within the NAc constitute less than 1%of the local neural population, yet they project throughout the NAc andprovide its only known cholinergic input. In this Example, anoptogenetic approach using a light-responsive chloride pump protein incombination with lanthanide-doped nanoparticles is used to block actionpotential firing in these cells, with both high temporal resolution andhigh cell-type specificity. To express microbial opsin genesspecifically in cholinergic interneurons, a transgenic mouse lineexpressing Cre recombinase is employed under the cholineacetyltransferase (ChAT) promoter. A Cre-inducible adeno-associatedvirus (AAV) vector carrying a yellow-light gated third-generationchloride pump halorhodopsin (eNpHR3.0) gene fused in-frame with codingsequence for enhanced yellow fluorescent protein (eYFP) isstereotactically injected.

Specifically, mice are anesthetized and then placed in a stereotactichead apparatus. Surgeries are performed on 4-6 week old mice andophthalmic ointment is applied throughout to prevent the eyes fromdrying. A midline scalp incision is made followed by a craniotomy, andthen AAV vector is injected with a 10 μl syringe and a 34 gauge metalneedle. The injection volume and flow rate (1 μl at 0.15 μl/min) arecontrolled by an injection pump. Each NAc receives two injections(injection 1: AP 1.15 mm, ML 0.8 mm, DV −4.8 mm; injection 2: AP 1.15mm, ML 0.8 mm, DV −4.2 mm). The virus injection and fiber position arechosen so that virtually the entire shell is stimulated.

Next, before withdrawing the needle, NaYF₄:Yb/Er/Gd, nanoparticles areinjected into the Nac. Concentrations of 3.4, 8.5, or 17 nmoles ofNaYF4:Yb/Er/Gd, nanoparticles are used. After injection of both the AAVvector and the lanthanide-doped nanoparticles is complete, the needle isleft in place for 5 additional minutes and then very slowly withdrawn.

Following a recovery period, the mice are again anesthetized, the skullsof the mice are thinned and an NIR source of electromagnetic radiationis placed adjacent to the thinned skull-region. Simultaneous NIRstimulation and extracellular electrical recording are performed basedon methods described previously using optical stimulation (Gradinaru etal., J. Neurosci., 27, 14231-14238 (2007)). The electrode consists of atungsten electrode (1 MΩ; 0.005 in; parylene insulation) with the tip ofthe electrode projecting beyond the fiber by 300-500 μm. The electrodeis lowered through the NAc in approximately 100 μm increments, andNIR-upconverted optical responses are recorded at each increment.Signals are amplified and band-pass filtered (300 Hz low cut-off, 10 kHzhigh cut-off) before digitizing and recording to disk. At each site, 5stimulation repetitions are presented and saved.

The examples, which are intended to be purely exemplary of the inventionand should therefore not be considered to limit the invention in anyway, also describe and detail aspects and embodiments of the inventiondiscussed above. The foregoing examples and detailed description areoffered by way of illustration and not by way of limitation. Allpublications, patent applications, and patents cited in thisspecification are herein incorporated by reference as if each individualpublication, patent application, or patent were specifically andindividually indicated to be incorporated by reference. In particular,all publications cited herein are expressly incorporated herein byreference for the purpose of describing and disclosing compositions andmethodologies which might be used in connection with the invention.Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

What is claimed is:
 1. A method to depolarize the plasma membrane of aneural cell in an individual comprising: (a) placing a plurality oflanthanide-doped nanoparticles in proximity to the neural cell, whereinthe nanoparticles comprise NaYF₄:Yb/X/Gd, wherein X is Erbium (Er),Thulium (Tm), or Er/Tm, and wherein the nanoparticles comprise anantibody or fragment thereof; and (b) exposing the plurality ofnanoparticles to electromagnetic radiation in the infrared (IR) or nearinfrared (NIR) spectrum, wherein the electromagnetic radiation in the IRor NIR spectrum is upconverted into light in the visible spectrum by thenanoparticles, and wherein a light-responsive opsin is expressed on theplasma membrane of the neural cell and activation of the opsin by thelight in the visible spectrum induces the depolarization of the plasmamembrane.
 2. The method of claim 1, wherein the light-responsive opsincomprises an amino acid sequence having at least 85% amino acid sequenceidentity to SEQ ID NO:5 or
 8. 3. The method of claim 1, wherein thelight-responsive opsin comprises an amino acid sequence having at least85% amino acid sequence identity to SEQ ID NO:6, 7, 9, 10, or
 11. 4. Themethod of claim 1, wherein the electromagnetic radiation in the IR orNIR spectrum is upconverted into light having a wavelength of about 450nm to about 550 nm.
 5. The method of claim 1, wherein theelectromagnetic energy in the IR or NIR spectrum is upconverted intolight having a wavelength corresponding to red, yellow, amber light,green light, or blue light.
 6. The method of claim 1, wherein theindividual is: (a) a non-human animal; or (b) a human.
 7. The method ofclaim 1, wherein the neural cell is a neural cell in the central nervoussystem; or in the peripheral nervous system.
 8. The method of claim 1,wherein the antibody or fragment thereof is conjugated to thenanoparticle.
 9. The method of claim 1, wherein the antibody or fragmentthereof specifically targets the neural cell.
 10. A method to depolarizethe plasma membrane of a neural cell in an individual comprising: (a)administering a polynucleotide encoding a light-responsive opsin to anindividual, wherein the light-responsive protein is expressed on theplasma membrane of a neural cell in the individual and the opsin iscapable of inducing membrane depolarization of the neural cell whenilluminated with light; (b) administering a plurality oflanthanide-doped nanoparticles in proximity to the neural cell, whereinthe nanoparticles comprise NaYF₄:Yb/X/Gd, wherein X is Erbium (Er),Thulium (Tm), or Er/Tm, and wherein the nanoparticles comprise anantibody or fragment thereof; and (c) exposing the plurality ofnanoparticles to electromagnetic radiation in the infrared (IR) or nearinfrared (NIR) spectrum, wherein the electromagnetic radiation in the IRor NIR spectrum is upconverted into light in the visible spectrum andthe activation of the opsin by the light in the visible spectrum inducesthe depolarization of the plasma membrane.
 11. The method of claim 10,wherein the light-responsive protein comprises an amino acid sequencehaving at least 85% amino acid sequence identity to SEQ ID NO:5, 6, 7,8, 9, 10, or
 11. 12. The method of claim 10, wherein the antibody orfragment thereof is conjugated to the nanoparticle.
 13. The method ofclaim 10, wherein the antibody or fragment thereof specifically targetsthe neural cell.
 14. A method to hyperpolarize the plasma membrane of aneural cell in an individual comprising: (a) placing a plurality oflanthanide-doped nanoparticles in proximity to the neural cell, whereinthe nanoparticles comprise NaYF₄:Yb/X/Gd, wherein X is erbium (Er),thulium (Tm), or Er/Tm, and wherein the nanoparticles comprise anantibody or fragment thereof; and (b) exposing the plurality ofnanoparticles to electromagnetic radiation in the infrared (IR) or nearinfrared (NIR) spectrum, wherein the electromagnetic radiation in the IRor NIR spectrum is upconverted into light in the visible spectrum by thenanoparticles, and wherein a light-responsive opsin is expressed on theplasma membrane and activation of the opsin by the light in the visiblespectrum induces the hyperpolarization of the plasma membrane.
 15. Themethod of claim 14, wherein the light-responsive opsin comprises anamino acid sequence having at least 85% amino acid sequence identity toSEQ ID NO:1 or SEQ ID NO:4.
 16. The method of claim 14, wherein theelectromagnetic energy in the IR or NIR spectrum is upconverted intolight having a wavelength of about 450 nm to about 550 nm.
 17. Themethod of claim 14, wherein the electromagnetic energy in the IR or NIRspectrum is upconverted into light having a wavelength corresponding tored, yellow, amber light, green light, or blue light.
 18. The method ofclaim 14, wherein the individual is: (a) a non-human animal; or (b) ahuman.
 19. The method of claim 14, wherein the neural cell is a neuralcell in the: (a) central nervous system; or (b) peripheral nervoussystem.
 20. The method of claim 14, wherein the antibody or fragmentthereof is conjugated to the nanoparticle.
 21. The method of claim 14,wherein the antibody or fragment thereof specifically targets the neuralcell.
 22. A method to hyperpolarize the plasma membrane of a neural cellin an individual comprising: (a) administering a polynucleotide encodinga light-responsive opsin to an individual, wherein the light-responsiveprotein is expressed on the plasma membrane of a neural cell in theindividual and the opsin is capable of inducing membranehyperpolarization of the neural cell when illuminated with light; (b)administering a plurality of lanthanide-doped nanoparticles in proximityto the neural cell, wherein the nanoparticles comprise NaYF₄:Yb/X/Gd,wherein X is Erbium (Er), Thulium (Tm), or Er/Tm, and wherein thenanoparticles comprise an antibody or fragment thereof; and (c) exposingthe plurality of nanoparticles to electromagnetic radiation in theinfrared (IR) or near infrared (NIR) spectrum, wherein theelectromagnetic radiation in the IR or NIR spectrum is upconverted intolight in the visible spectrum and the activation of the opsin by thelight in the visible spectrum induces the hyperpolarization of theplasma membrane.
 23. The method of claim 22, wherein thelight-responsive opsin comprises an amino acid sequence having at least85% amino acid sequence identity to SEQ ID NO:1 or SEQ ID NO:4.
 24. Themethod of claim 22, wherein the antibody or fragment thereof isconjugated to the nanoparticle.
 25. The method of claim 22, wherein theantibody or fragment thereof specifically targets the neural cell.
 26. Asystem comprising: a) lanthanide-doped nanoparticles comprisingNaYF₄:Yb/X/Gd, wherein X is Erbium (Er), Thulium (Tm), or Er/Tm, andwherein the nanoparticles further comprise an antibody or fragmentthereof; b) a nucleic acid comprising a nucleotide sequence encoding alight-responsive polypeptide; and c) a source of infrared or nearinfrared electromagnetic radiation.
 27. The system of claim 26, whereinthe antibody or fragment thereof is conjugated to the nanoparticle. 28.The system of claim 26, wherein the antibody or fragment thereofspecifically targets a neural cell.